One long-standing limitation on the resolution achievable in optical-regime microscopy has been the diffraction limit of the illuminating light. Details having size equal to or less than half the wavelength of the illuminating light are beneath the resolving powers of traditional optical microscopes. Thus, details in the range of 250 nanometers or smaller cannot be resolved using conventional focal optical microscopy.
In recent years, a solution has been provided to this problem by near-field scanning optical microscopy (NSOM). This technology permits optical resolution below the normal diffraction limit by using a fiber optic light guide to direct illuminating light from an aperture with dimensions of less than half the wavelength of the light (typically about 20-100 nanometers) at the light guide tip onto an equally small area of the specimen. The tip is located very close to the specimen, so that the specimen lies in the near-field region of the light emanating from the tip, and diffraction limitations play no role. The near field is generally the area within less than a wavelength or so of the aperture from which the illuminating light emanates.
The illumination is measured by collecting either reflected, transmitted or fluorescent light, and that measurement yields a single pixel of the microscopic image. The sample is typically scanned in a raster pattern across the surface of the specimen to yield an image having a pixel resolution on the order of the tip dimensions, namely about 20-50 nanometers, which lies below the normal diffraction barrier. Some examples of an NSOM are disclosed in U.S. Pat. No. 4,917,462 to Lewis et al., and in U.S. Pat. Nos. 5,272,330 and 5,286,970 to Betzig, et al.
U.S. Pat. No. 4,917,462 to Lewis et al. discloses an NSOM apparatus involving a metal-coated glass pipette having a thin tip. Lewis teaches the application of an electrical potential between the pipette and the stage, resulting in a measurable current to provide a feedback signal used to determine the distance between aperture and object, see col. 11, 1. 27-52, although he also mentions the possibility of using light for feedback, col. 11, line 53 et seq. Lewis teaches general epi-fluorescence illumination of the specimen, although he also mentions the possibility of providing light through the pipette to the specimen.
In NSOM, it may be preferable to use a fiber optic light guide with a fine tip in order to provide more light directly to the specimen than could be provided through a pipette. See U.S. Pat. Nos. 5,272,330 and 5,286,970 to Betzig, et al. Desirably, illuminating light will be directed from a very small aperture at the light guide tip onto a very small area of a nearby specimen.
A significant difficulty in NSOM has been determining when the fiber optic light guide tip has reached a location close enough to the specimen that the specimen surface lies within the near-field of the illuminating light. Furthermore, the tip must be maintained at a constant elevation above the surface being scanned. Various feedback techniques have been developed in the field to maintain this tip-sample registration. Generally, the light guide tip is moved toward the specimen until the tip is in the immediate vicinity of the specimen. Then, using a "shear-force" feedback method, the tip is held within 5-20 nanometers of the sample. The scanning is then performed by mechanically moving the sample under the tip and registering light reflectance on transmission values.
These techniques generally work well for relatively dry, hard and flat samples, but do not work well with biological specimens, which are soft, have greater topological variation, and do not advantageously yield mechanical and electrical signals which can be measured to determine the proximity of the tip to the specimen surface. Furthermore, these techniques tend to have a destructive effect on the biological material.
For example, using "shear force" feedback, the tip is vibrated side-to-side at its resonant frequency as it is brought down toward the surface of the specimen. A phase shift or damping of the vibration caused by forces of interaction between the tip and the sample is used as a measure of separation to hold the tip at a constant distance from the sample. This technique works well for dry, relatively flat surfaces. Unfortunately, such a technique, if used on living cells in solution, can result in damage to biological cell membranes from the side-to-side vibration. In U.S. Pat. No. 5,354,985 to Quate, a cantilever light guide is disclosed which determines proximity to the specimen surface by measuring changes in the resonant vibration frequency of the cantilever. As the cantilever aperture tip approaches the surface of the specimen, Van der Waals forces influence the resonant frequency. In an alternative embodiment disclosed in Quate, the cantilever is brought into contact with the surface by measuring deflection of the cantilever from a rest position by the force of the surface. The cantilever is then dragged over the surface while maintaining a constant force on it.
The microscope of Quate works well with well-defined, crystalline surfaces of relatively homogenous composition. However, biological specimens such as cells have surfaces littered with structures which often extend relatively far from the cell membrane. The apparatus of Quate is not well-adapted to measure proximity to the cell membrane because these structures also impart Van der Waals forces affecting the resonant frequency of the cantilever. Furthermore, the surface of the cell is composed of a wide variety of molecules with varying Van der Waal attraction forces. No consistent or useful scanning of a living biological specimen can be carried out using the apparatus of Quate.
U.S. Pat. No. 5,340,981 to De Fornel et al. discloses a means of bringing a NSOM within the near-field of a specimen using the electromagnetic coupling between the incident illumination radiation and the reflected radiation. The invention is best suited for coupling the modes of the reflected light with the illumination light when the surface being scanned is well-defined and preferably near-crystalline Biological cells with their highly heterogeneous surface structures do not present the kind of surfaces from which the reflective modes used in De Fornel can be advantageously obtained. Furthermore, the form of microscopy described by DeFornell specifically relates to reflection and not fluorescence microscopy.
Another genre of microscopes related to NSOMs is scanning tunneling microscopes. Again, the technique used in the scanning tunneling microscope is suited for flat, dry specimens, but not for living biological cells. Scanning tunneling microscopy utilizes tunneling electrons as a signal source to indicate distance between the probe tip and the specimen. As the tip approaches the specimen, the tunneling phenomenon of electrons becomes measurable and increases with increasing proximity. However, an electrically conductive specimen is required for this kind of microscopy.
In the field of biological microscopy, it is desirable to achieve nanometer-scale optical resolution in order to study cellular metabolism, and in particular cell membrane metabolism. Metabolism is studied both in terms of the spatial distribution of molecular structures and also in terms of the time-variation of molecular concentrations. An example of the need for such optical resolution is the study of ion channels in the cell membrane. Such membrane structures have dimensions on the order of 1-30 nanometers. The flow of ions across the membrane into the cell is desirably monitored by measuring the fluorescence of an indicator activated in the presence of the ions. Traditional microscopy produces a volume-averaged measurement, since the contribution to fluorescence by indicators just below the cell membrane is swamped by the dye throughout the cell. If it were possible to map fluorescence locally at tens-of-nanometers resolution, the volume-averaged effect would be avoided, and it would be possible to map the distribution of functional ion channels and potentially individual molecules over the cell membrane.
In view of the above prior techniques, it is apparent there are obstacles to sub-diffraction limit microscopic resolution in imaging living biological cells. The techniques described above generally require the specimen to be flat, dry, well-defined at the surface, hard or crystalline. When practiced on living biological cells, the techniques either yield inaccurate results or may damage a living biological cell.
A further problem in optical microscopy of biological specimens has been that virtually all of the above-described techniques are designed to measure only the surface topology of a specimen, usually for fractures or inconsistencies in crystalline structure. None of the above techniques is suitable for monitoring metabolic activity through time, for monitoring cortical concentrations of fluorescent-tagged molecules at the cell membrane, or for detecting fluorescence-labelled molecules associated with the cell membrane.
What is needed is a means for generating accurate microscopic images of living biological structures below the 250 nanometer resolution limitation of conventional optical microscopy, and preferably in the range of tens of nanometers More importantly, such a needed microscopic apparatus must not damage the biological structures, and must be able to provide an accurate image of metabolic activity without interfering with such activity.